1. Field of the Invention
The present invention relates to microscopy imaging apparatus and an imaging method, for generating an image which generally contains only in-focus detail and a method thereof. The present invention is suited to generating images which may be used to create in-focus three-dimensional images of volume structures.
2. Related Art
With a conventional light microscope three-dimensional structures cannot be successfully imaged. The resultant image consists of a sharp image of an in-focus region of a three-dimensional structure as well as defocused images of the structures above and below the in-focus region. A conventional light microscope is unable to reject out-of-focus detail.
Confocal microscopes have been developed which optically section a three-dimensional structure to provide in-focus images of individual layers or strata of the structure which can be subsequently combined to form an in-focus three-dimensional image of the complete volume structure. Unfortunately, the light budget of confocal microscopes is generally poor when incoherent light sources are used. Laser scanning confocal microscopes can achieve a very shallow depth of focus, but require expensive apparatus and an illuminating/imaging pinhole through which the laser light must be focused.
In U.S. Pat. No. 5,381,236 an optical sensor is described which is used to determine the range (distance) of individual features of a three-dimensional structure. The sensor has a periodic patterned light source which illuminates the structure and is reversible (i.e. the pattern is phase shifted 180xc2x0). An array of detector elements which are aligned to the pattern of the light source is used to detect an image of the pattern and the reversal of the pattern illuminating the structure. As the pattern will only be imaged well on those parts of the structure which are themselves in focus, this enables the range (distance) of in-focus parts of the structure to be determined. The apparatus and method described in U.S. Pat. No. 5,381,236 has the disadvantage that in order to work the individual elements of the detector must be exactly aligned with and matched to the pattern of the light source. In practice this has been found to be almost impossible to achieve.
The present invention seeks to provide microscopy imaging apparatus and an imaging method to produce images which, in a similar way to confocal images, comprise substantially only in-focus detail and which can be used to create a three-dimensional image of a structure through optical sectioning of the structure. The present invention achieves the optical sectioning without the need for precise alignment or matching of detector and pattern components and provides at the same time a favourable light budget.
In one aspect, the present invention provides a method of generating an image of a specimen from three or more recorded images of the specimen, each recorded image having superimposed thereon a substantially periodic spatial pattern, the spatial phase of the pattern in at least three recorded images of the specimen being different; the method comprising grouping the recorded images into pairs of images and calculating the square root of (the sum of the squares of the differences between the recorded images in each pair of images), thereby to remove the spatial pattern and obtain an in-focus image.
In another aspect, the present invention provides a method of generating an image of a specimen comprising the steps of illuminating the specimen with a light source; generating a substantially periodic spatial pattern on the specimen; recording a first image of the specimen; altering the spatial phase of the pattern on the specimen and recording a second image of the specimen; repeating at least once more the step of altering the spatial phase of the pattern on the specimen and recording a third image of the specimen, the spatial phase of the pattern in at least three recorded images of the specimen being different; and analysing the three or more recorded images of the specimen to remove the spatial pattern from the images thereby to generate an optically sectioned image of the specimen.
While the identified prior art relies on a matched detector grid aligned to stringent requirements with the mask pattern, the present invention is of advantage in that the need for such a matched detector grid is eliminated. Processing of image data is simple and the invention enables production of optically sectioned images from a conventional microscope in real time.
The analysis suitably comprises grouping the recorded images into pairs of images and calculating the square root of the sum of the squares of the differences between the recorded images in each pair of images, thereby to remove the spatial pattern and obtain an in-focus image. In one embodiment of the invention the analysis comprises removing the pattern using the formula:   I  =                    ∑                  n          ,          m                    ⁢                        (                                    I              n                        -                          I              m                                )                2            
wherein the recorded images are In,m and the in-focus image is I. Thus, where there are three recorded images the in-focus image can be calculated from the formula:
I={square root over ((I1xe2x88x92I2+L )2+L +(I1xe2x88x92I3+L )2+L +(I2xe2x88x92I3+L )2+L )}
Where there are four recorded images the analysis can use different symmetrical pairs of images, such as (i) images 1 and 2; 2 and 3; 3 and 4; and 4 and 5 or (ii) images 1 and 3; and 2 and 4, and thus the formula can be:
xe2x80x83I={square root over ((I1xe2x88x92I3+L )2+L +(I2xe2x88x92I4+L )2+L )}
or:   I  =                              (                                    I              1                        -                          I              2                                )                2            +                        (                                    I              2                        -                          I              3                                )                2            +                        (                                    I              3                        -                          I              4                                )                2            +                        (                                    I              4                        -                          I              1                                )                2            
Following this principle, the recorded images are separated into pairs of images, or into symmetrical subsets of pairs of images, and analyzed according to the invention. Thus, it is not necessary for all possible pairs of images to be used in calculating the in-focus image, and the in-focus image can be calculated from just one subset of pairs.
In a preferred embodiment the method of the invention is repeated at different focal positions to produce an in-focus three-dimensional image of the specimen. In a specific embodiment of the invention, described in detail below, a three-dimensional image of the surface texture of a specimen is thereby obtained.
The spatial phase of the pattern may be altered continuously or in discrete steps. Where the spatial phase is altered continuously, the recorded images of the specimen are integrated over a predetermined time period. It has been calculated and also confirmed in practice that high quality images are obtained when the spatial phase of the pattern is continuously altered. Thus, a wide range of means for altering the spatial phase are of application to the present invention.
In a further aspect, the present invention provides a method of processing image data of at least three images of the same specimen, the images having a substantially periodic pattern superimposed thereon, the spatial phases of the pattern on three images being different, comprising analysing the data so as to generate a composite image, free of the pattern.
In a still further aspect, the present invention provides microscopy imaging apparatus comprising a light source; patterning means for generating a substantially periodic spatial pattern; focusing means for focusing light from the light source on a specimen and generating the pattern on the specimen; phase shift means for adjusting the spatial phase of the pattern generated on the specimen; a detector for detecting images of the specimen; and an analyzer having means for analysing images of the specimen, the spatial phase shift of the pattern being different in at least three images, and means for removing the spatial pattern from the three images of the specimen thereby to generate an optically sectioned image of the specimen.
Preferably, the patterning means is in the form of a mask having one-dimensional local periodicity and the mask pattern is projected onto the specimen. For example, the mask may be a linear grating. The mask may also be a circular mask containing a spiral grating. In this latter case the pattern is conveniently projected onto the specimen by illuminating the specimen through a portion of the grating located towards or at the edge of the circular mask; for in this portion, the spiral grating approximates to a grating of parallel lines. An advantage of this spiral grating is that continuous movement of the grating is achieved by rotation of the mask. Alternatively, where a coherent light source is employed, the patterning means may be provided by a second coherent light source so arranged to interfere with the light from the first light source.
In a further aspect, the present invention provides apparatus adapted to modify a conventional microscope, comprising patterning means for generating a substantially periodic spatial pattern, phase shift means for adjusting the spatial phase of the pattern and an analyzer having means for analysing images of a specimen on which the spatial pattern has been generated, the spatial phase shift of the pattern being different in at least three images, and means for removing the spatial pattern from the three images thereby to generate an optically sectioned image of the specimen.
In a further embodiment, the present invention provides a method of adapting a conventional microscope to produce optically sectioned images of a specimen, the method comprises introducing patterning means into the optical system of the microscope for generating a substantially periodic spatial pattern on the specimen; providing spatial phase shift means for adjusting the spatial phase of the pattern to produce at least three different spatially phase shifted patterns on the specimen; and providing an analyzer having means for analysing at least three separate images of the specimen, each with a different spatial phase shift of the pattern, and means for removing the spatial pattern from the images of the specimen thereby to generate optically sectioned image of the specimen.
It is thus an advantage of the present invention that a conventional microscope, a ubiquitous piece of laboratory equipment, may readily be converted so as to be capable of providing optically sectioned images.
The present invention also provides a method of processing image data of at least three images of the same specimen, the images having a substantially periodic pattern superimposed thereon, the spatial phases of the pattern on three images being different, comprising analysing the data so as to generate a composite, from said image, free of the pattern.